ZIKV infection effects changes in gene splicing, isoform composition and lncRNA expression in human neural progenitor cells

Abstract

Background: The Zika virus (ZIKV) is a mosquito-borne flavivirus that causes microcephaly and Guillain-Barré syndrome in infected individuals. To obtain insights into the mechanism of ZIKV infection and pathogenesis, we analyzed the transcriptome of ZIKV infected human neural progenitor cells (hNPCs) for changes in alternative splicing (AS), gene isoform (ISO) composition and long noncoding RNAs (lncRNAs) expression.

Methods: We analyzed differentially expressed lncRNAs, AS, ISO from RNA-seq data in ZIKV infected hNPCs.

Results: We obtained 149 differentially expressed lncRNAs, including potential viral targets to modulate cellular processes such as cell cycle, apoptosis and immune response. The infection induced 262 cases of AS occurring in 229 genes, which were enriched in cell death, RNA processing, transport, and neuron development. Among 691 differentially expressed ISOs, upregulated ISOs were enriched in signaling, regulation of transcription, and amino acid biosynthesis, while downregulated ISOs were mostly enriched in cell cycle. Importantly, these analyses revealed specific links between ZIKV induced changes in cellular pathways and the type of changes in the host transcriptome, suggesting important regulatory mechanisms.

Conclusions: Our analyses revealed candidate lncRNAs, AS events and ISOs which may function in ZIKV infection induced cell cycle disruption, apoptosis and attenuation of neurogenesis, and shed light on the roles of lncRNAs, AS and ISOs in virus-host interactions, and would facilitate future studies of ZIKV infection and pathogenesis.

Keywords: Alternative splicing; Gene isoform; ZIKV; hNPC; lncRNA.

Fig. 1

Novel lncRNAs in ZIKV infected human neuro progenitor cells. a. The number of candidate novel lncRNAs; b. The novel lncRNAs represent smaller the number of exon than PCGs on average; c. The PCGs represent higher expression level than novel lncRNAs on average; d. The novel lncRNAs show shorter length on average than PCGs .Wilcox.test, p value < 0.05

Fig. 2

Expression of lncRNAs in ZIKV infected cells. a, b. heatmap showed 92 annotated and 57 novel DE lncRNAs, respectively. c,d,e,f Visualization of two novel and annotated lncRNAs. g, h. expression level of the indicated lncRNAs. Each group consists of two biological replicates. *: FDR  0.05, **:FDR <0.01

Fig. 3

ZIKV infection induced alternative splicing of the cellular transcriptome. a. 262 significant differentially AS cases occurred 229 significant alternative splicing genes were detected, which falls into skipped exon, skipped multiple exon, alternative to 5′splicing site (AS5), alternative to 3′splicing site (AS3), alternative start (altstart), alternative end (altend), mutually exclusive and retained intron classes [16]; b. GO analysis of AS genes, the x-axis is the –log₁₀ ^{p value}, Black, gray, read, and blue denotes cell death, neuron development, transport, and RNA processing, respectively; c–k. Visualization of alternative splicing. Y-axis shows the number of mapped reads. c. FANCA: increased SE after infection; d. HRAS: increased exon after infction; e. HNRNPA2B1: reduced exon after infection; f PSPC1: reduced altend after infection

Fig. 4

Isoform changes in the ZIKV infected hNPCs transcriptome. a A gene with isoform changes of at least 2-fold (FPKM ≥ 1, FDR ≤ 0.05) was plotted. A Total of 691 significant isoform changes were found with 355 gene isoforms showing increase of expression and 336 showing reduction. X-axis shows uninfected hNPCs control, and Y-axis shows the amount of isoform expression after infection. Red dots indicate up-regulated gene isoforms and blue dots show reduced isoform expression; b. the overlapping genes of AS and ISO; c, d. GO analysis and pathway analysis of down-regulated isoforms, p value ≤ 0.05; e, f. GO analysis and pathway analysis of up-regulated isoforms, p value ≤ 0.05. g,h. expression level of E2F1, E2F2, CXB4, HEY1, TNFRSF10D, and BBC3. Each group consists of two biological replicates. *: FDR < 0.05, **:FDR 0.01. The x-axis of c/d/e/f graphs is the –log₁₀ ^{p value}

Fig. 5

ZIKV induced host responses and alterations of host transcriptome. Several DE lncRNAs, ISOs and PCGs were enriched in cell cycle, apoptosis, immune response, transcription regulation, amino acid biosynthesis, mTOR signaling pathway and p53 signaling pathway. Meanwhile, many of genes undergone AS involved in neuron development, RNA processing and transport. Red and green arrows denote downregulation and upregulation, respectively. The rectangle and oval stand for GO terms and pathways, respectively